Molecular serotyping of Escherichia coli O111:H8

Accurate Escherichia coli serotyping is critical for pathogen diagnosis and surveillance of non-O157 Shiga-toxigenic strains, however few laboratories have this capacity. The molecular serotyping protocol described in this paper targets the somatic and flagellar antigens of E. coli O111:H8 used in traditional serotyping, and can be performed routinely in the laboratory.     

Molecular Serotyping of Escherichia coli O26:H11

Serotyping is the foundation of pathogenic Escherichia coli diagnostics; however, few laboratories have this capacity. We developed a molecular serotyping protocol that targets, genetically, the same somatic and flagellar antigens of E. coli O26:H11 used in traditional serotyping. It correctly serotypes strains untypeable by traditional methods, affording primary laboratories serotyping capabilities.     

Identification and molecular epidemiology of Campylobacter coli isolates from human gastroenteritis, food, and animal sources by amplified fragment length polymorphism analysis and Penner serotyping

Campylobacter coli is an infrequently studied but important food-borne pathogen with a wide natural distribution. We investigated its molecular epidemiology by use of amplified fragment length polymorphism (AFLP)-based genotyping and Penner serotyping. Serotype reference strains and 177 Danish isolates of diverse origin identified by routine phenotyping as C. coli were examined. Molecular tools identified some 12% of field isolates as Campylobacter jejuni, emphasizing the need for improved identification methods in routine laboratories. Cluster analysis of AFLP profiles of 174 confirmed C. coli isolates revealed a difference in the distribution of isolates from pig and poultry (chicken, duck, turkey, and ostrich) species and indicated the various poultry species, but not pigs, to be likely sources of human C. coli infection. A poor correlation was observed between serotyping and AFLP profiling, suggesting that the former method has limited value in epidemiological studies of this species.     

Detection of Escherichia coli serogroups O26 and O113 by PCR amplification of the wzx and wzy genes

PCR-based assays for detecting enterohemorrhagic Escherichia coli serogroups O26 and O113 were developed by targeting the wzx (O-antigen flippase) and the wzy (O-antigen polymerase) genes found in the O-antigen gene cluster of each organism. The PCR assays were specific for the respective serogroups, as there was no amplification of DNA from non-O26 and non-O113 E. coli serogroups or from other bacterial genera tested. Using the PCR assays, we were able to detect the organisms in seeded apple juice inoculated at concentration levels as low as < or =10 CFU/ml. The O26- and O113-specific PCR assays can potentially be used for typing E. coli O26 and O113 serogroups; these assays will offer an advantage to food and environmental microbiology laboratories in terms of identifying these non-O157 serogroups by replacing antigen-based serotyping.     

Production, characterization, and application of antibodies against heat-labile type-I toxin for detection of enterotoxigenic Escherichia coli

Strains of enterotoxigenic Escherichia coli (ETEC) are responsible for significant rates of morbidity and mortality among children, particularly in developing countries. The majority of clinical and public health laboratories are capable of isolating and identifying Salmonella, Shigella, Campylobacter, and Escherichia coli O157:H7 from stool samples, but ETEC cannot be identified by routine methods. The method most often used to identify ETEC is polymerase chain reaction for heat-stable and heat-labile enterotoxin genes, and subsequent serotyping, but most clinical and public health laboratories do not have the capacity or resources to perform these tests. In this study, polyclonal rabbit and monoclonal mouse IgG2b antibodies against ETEC heat-labile toxin-I (LT) were characterized and the potential applicability of a capture assay was analyzed. IgG-enriched fractions from rabbit polyclonal and the IgG2b monoclonal antibodies recognized LT in a conformational shape and they were excellent tools for detection of LT-producing strains. These findings indicate that the capture immunoassay could be used as a diagnostic assay of ETEC LT-producing strains in routine diagnosis and in epidemiological studies of diarrhea in developing countries as enzyme linked immunosorbent assay techniques remain as effective and economical choice for the detection of specific pathogen antigens in cultures.     

A colony blot immunoassay to detect enteroinvasive Escherichia coli and Shigella in water samples

AIMS: The aim of the study was to develop a colony blot immunoassay to detect Shigella and enteroinvasive Escherichia coli (EIEC) in water. METHODS AND RESULTS: Spiked samples were filtered through nitrocellulose membranes. Colony prints on the filters were tested with a monoclonal antibody specific to IpaC, an antigen coded by the invasion plasmid of Shigella and EIEC. Invasive pathogens could be successfully detected with the technique, even in the presence of a large number of non-pathogenic bacterial cells. The method was significantly more sensitive in identifying pathogen-containing samples then the traditional culture-based approach. CONCLUSION: The IpaC-specific colony blot immunoassay is an inexpensive method for identifying the aetiological agents of bacillary dysentery in water samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The technique could be particularly useful in detecting enteroinvasive E. coli which often remains undetected by bio- and serotyping.     

Discrepancy between Penner serotyping and polymerase chain reaction fingerprinting of Campylobacter isolated from poultry and other animal sources

Thirty-four Campylobacter jejuni or coli strains, isolated from various livestock and darkling beetles from two Dutch poultry farms during different broiler production cycles, were subjected to Penner serotyping and polymerase chain reaction (PCR) fingerprint analysis. Ten different Penner serotypes were determined in the isolates. Visual scoring of the PCR fingerprints resulted in 14 clearly different profiles. Some strains with identical Penner serotypes exhibited different PCR fingerprints and conversely strains with different serotypes produced identical PCR fingerprints. Discrepancies between Penner serotyping and PCR fingerprinting were most obvious between isolates from different animal sources. Indications for the occurrence of genomic rearrangements were found. The inconsistency between serotyping and fingerprinting of Campylobacter strains suggests that conventional typing methods should be used in combination with fingerprinting if the epidemiological factors that contribute to Campylobacter colonization of live chickens are to be assessed reliably.     

Examination of retail chickens and sausages in Britain for vero cytotoxin-producing Escherichia coli.

Samples from chickens and pork sausages were examined for the presence of Vero cytotoxin-producing Escherichia coli by using DNA probes for the Vero cytotoxin genes. Hybridization was detected in 25% of the 184 sausage samples, but none of the chickens was positive. No E. coli O157:H7 strains were isolated, and serotyping showed that the Vero cytotoxin-producing E. coli strains belonged to eight different O serogroups and that six strains had an unidentifiable O antigen.     

Examination of retail chickens and sausages in Britain for vero cytotoxin-producing Escherichia coli

Samples from chickens and pork sausages were examined for the presence of Vero cytotoxin-producing Escherichia coli by using DNA probes for the Vero cytotoxin genes. Hybridization was detected in 25% of the 184 sausage samples, but none of the chickens was positive. No E. coli O157:H7 strains were isolated, and serotyping showed that the Vero cytotoxin-producing E. coli strains belonged to eight different O serogroups and that six strains had an unidentifiable O antigen.     

Growth of Escherichia coli in a pulp and cardboard mill

The coliform flora of a pulp and cardboard mill that uses birch as the raw material and ammonium sulphate as the process chemical was studied. Escherichia coli was observed to multiply in the mill. It persisted as the dominant thermotolerant coliform in the effluent. Klebsiellae were encountered among total coliforms only. The E. coli strains isolated had the biochemical characteristics and maximum growth temperatures typical to the species. However, serotyping and hemolysin test differentiated these strains from pathogenic and fecal E. coli.     

Serotyping of Vibrio anguillarum.

A serotyping scheme based on the detection of O antigens by slide agglutination in fish-pathogenic strains of Vibrio anguillarum is presented. Over a period of 5 years 270 Vibrio strains from feral and cultured fish, 189 strains from the environment, and 36 strains from invertebrates were collected. The strains were divided into 10 distinct serotypes (O1 through O10). More than 90% of the fish-pathogenic strains, but only 40% of the environmental strains, were typable; 71% of the strains isolated from cultured rainbow trout were serotype O1, whereas 78% of the strains isolated from feral fish were serotype O2. No dominating environmental serotype was found. A serotyping system for V. anguillarum is proposed. A total of 90 strains received from culture collections and laboratories in different countries were typed according to the present system.     

Serotyping of Vibrio anguillarum

A serotyping scheme based on the detection of O antigens by slide agglutination in fish-pathogenic strains of Vibrio anguillarum is presented. Over a period of 5 years 270 Vibrio strains from feral and cultured fish, 189 strains from the environment, and 36 strains from invertebrates were collected. The strains were divided into 10 distinct serotypes (O1 through O10). More than 90% of the fish-pathogenic strains, but only 40% of the environmental strains, were typable; 71% of the strains isolated from cultured rainbow trout were serotype O1, whereas 78% of the strains isolated from feral fish were serotype O2. No dominating environmental serotype was found. A serotyping system for V. anguillarum is proposed. A total of 90 strains received from culture collections and laboratories in different countries were typed according to the present system.     

Extraintestinal Escherichia coli isolations from SIDS cases and other cases of sudden death in Victoria, Australia

This investigation is an extension of previous studies on the possible role of intestinal Escherichia coli in sudden infant death syndrome (SIDS) to include the isolation of extraintestinal E. coli. The lungs of 52 and the blood of 144 SIDS infants were cultured and isolates were investigated. E. coli was isolated from about a quarter of post-mortem lung samples and about 15% of blood samples from SIDS infants. The isolates were subjected to microbiological studies, including serotyping and haemolysin assays. The majority were found to belong to serogroups commonly associated with bacteraemia. These results may indicate that extraintestinal E. coli plays a role in SIDS.     

Atypical Escherichia coli in streams.

In the examination of stream waters for fecal coliforms, pale yellow colonies regularly appeared on m-FC broth base medium plates. The yellow colonies may comprise 70% more of the colonies of an m-FC plate. More than 80% of these colonies were identified as Escherichia coli by the API 20E identification system and by serotyping. The atypical yellow E. coli strains were not environmentally stressed E. coli since the atypical colonies continued to be yellow on m-FC medium after growth in a nonselective medium. However, 50% of the atypical E. coli strains were o-nitrophenyl-beta-D-galactopyranoside positive, and 20% produced gas in EC medium at 44.5 degrees C. Failure to consider these atypical E. coli strain in water quality analyses could lead to a significant error in the estimation of water quality in some instances.     

Atypical Escherichia coli in streams

In the examination of stream waters for fecal coliforms, pale yellow colonies regularly appeared on m-FC broth base medium plates. The yellow colonies may comprise 70% more of the colonies of an m-FC plate. More than 80% of these colonies were identified as Escherichia coli by the API 20E identification system and by serotyping. The atypical yellow E. coli strains were not environmentally stressed E. coli since the atypical colonies continued to be yellow on m-FC medium after growth in a nonselective medium. However, 50% of the atypical E. coli strains were o-nitrophenyl-beta-D-galactopyranoside positive, and 20% produced gas in EC medium at 44.5 degrees C. Failure to consider these atypical E. coli strain in water quality analyses could lead to a significant error in the estimation of water quality in some instances.     

Analysis of environmental Escherichia coli isolates for virulence genes using the TaqMan PCR system

AIMS: To assess the presence of virulence genes in environmental and foodborne Escherichia coli isolates using the TaqMan PCR system. METHODS AND RESULTS: Three TaqMan pathogen detection kits called O157:H7, StxI and StxII were used to investigate the presence of virulence genes in Escherichia coli isolates. All 54 foodborne E. coli O157:H7 isolates showed expected results using these kits. Ninety (15%) of 604 environmental isolates gave positive amplification with an O157:H7-specific kit. TaqMan PCR amplification products from these 90 isolates were analysed by agarose gel electrophoresis, and 90% (81 of 90) of the environmental samples contained the expected PCR product. Sixty-six of these 90 were chosen for serotyping tests and only 35% (23 of 66) showed agglutination with both anti-O157 and anti-H7 antibodies. Further ribotyping of 16 sero-positive isolates in an automated Riboprinter did not identify these to be O157:H7. Multiplex PCR with primers for eaeA, stxI and stxII genes was used to confirm the TaqMan results in 10 selected environmental isolates. CONCLUSIONS: All three TaqMan pathogen detection kits were useful for virulence gene analysis of prescreened foodborne O157:H7 isolates, while the O157:H7-specific kit may not be suitable for virulence gene analysis of environmental E. coli isolates, because of high false positive identification. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to rapidly identify the presence of pathogenic E. coli in food or environmental samples is essential to avert outbreaks. These results are of importance to microbiologists seeking to use TaqMan PCR to rapidly identify pathogenic E. coli in environmental samples. Furthermore, serotyping may not be a reliable method for identification of O157:H7 strains.     

Genetic characteristics and antimicrobial resistance of Escherichia coli from Japanese macaques (Macaca fuscata) in rural Japan

Escherichia coli was isolated from wild and captive Japanese macaques (Macaca fuscata) to investigate the risk of zoonotic infections and the prevalence of antimicrobial-resistant Escherichia coli in the wild macaque population in Shimokita Peninsula, a rural area of Japan. We collected 265 fresh fecal samples from wild macaques and 20 samples from captive macaques in 2005 and 2006 for E. coli isolation. The predominant isolates were characterized by serotyping, virulence gene profiling, plasmid profiling, pulsed-field gel electrophoresis (PFGE), and microbial sensitivity tests. In total, 248 E. coli strains were isolated from 159 fecal samples from wild macaques, and 42 E. coli were isolated from 17 samples from captive macaques. None of the virulence genes eae, stx, elt, and est were detected in any of the isolates. The relatedness between wild- and captive-derived isolates was low by serotyping, PFGE, and plasmid profiling. Serotypes O8:H6, O8:H34, O8:H42, O8:HUT, O103:H27, O103:HNM, and OUT:H27 were found in wild macaque feces; serotypes O157:H42 and O119:H21 were recovered from captive macaques. O-and H-serotypes of the 26 isolates were not typed by commercial typing antisera and were named OUT and HUT, respectively. Twenty-eight isolates had no flagellar antigen, and their H-serotypes were named HNM. Similarity of PFGE patterns between wild-derived isolates and captive-derived isolates was <70%. No plasmid profile was shared between wild-derived and captive-derived isolates. The prevalence of antimicrobial-resistant E. coli was 6.5% (n=62) in wild macaques, and these isolates were resistant to cephalothin. We conclude that wild Japanese macaques in Shimokita Peninsula were unlikely to act as a reservoir of pathogenic E. coli for humans and that antimicrobial-resistant E. coli in wild macaques may be derived from humans.     

Identification and Molecular Epidemiology of Campylobacter coli Isolates from Human Gastroenteritis, Food, and Animal Sources by Amplified Fragment Length Polymorphism Analysis and Penner Serotyping

Campylobacter coli is an infrequently studied but important food-borne pathogen with a wide natural distribution. We investigated its molecular epidemiology by use of amplified fragment length polymorphism (AFLP)-based genotyping and Penner serotyping. Serotype reference strains and 177 Danish isolates of diverse origin identified by routine phenotyping as C. coli were examined. Molecular tools identified some 12% of field isolates as Campylobacter jejuni, emphasizing the need for improved identification methods in routine laboratories. Cluster analysis of AFLP profiles of 174 confirmed C. coli isolates revealed a difference in the distribution of isolates from pig and poultry (chicken, duck, turkey, and ostrich) species and indicated the various poultry species, but not pigs, to be likely sources of human C. coli infection. A poor correlation was observed between serotyping and AFLP profiling, suggesting that the former method has limited value in epidemiological studies of this species.